Difference between revisions of "Part:BBa K3198002"

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RES

Sequence and Features

Description

This part contains the toxin component of a type II toxin-antitoxin (TA) system. RES is orthologous to E. coli and was named after three conserved amino acids, arginine (R), glutamate (E) and serine (S).

Usage

Team NUS Singapore 2019 has added a new biobrick (BBa_K3198002) into the iGEM repository this year. This biobrick was found to possess the ability to inhibit cell growth by acting as a NAD+ glycohydrolase (NADase). For this reason, team NUS Singapore 2019 used this biobrick as part of their sleep-wake module to control the growth of E. coli by inducing metabolite-mediated dormancy in these cells.

Biology

This part originated from the RES-Xre locus from Photorhabdus luminescens and other bacterial species. The activation of this toxin in vivo has been shown to deplete intracellular NAD+ levels, subsequently resulting in the inhibition of global macromolecular biosynthesis as well as cell growth in Escherichia coli.

Characterization

Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198002 expression would have resulted in reduced growth, supported by reduced OD600 which is an indicator of biomass.

To test this hypothesis, BBa_K3198002 which was originally provided by Professor Brodersen was placed under an arabinose-inducible promoter. Arabinose concentrations of 0.2% and 0.4% were explored to determine their effect on the growth of native MG1655.

Characterization of cells transformed with this plasmid was performed using microplate reader at 37°C for 20h continuously. The cells started off with an OD600 of 0.1 at 0h before they were treated with both 0.2% and 0.4% arabinose at 1h to induce the expression of BBa_K3198002.

The team observed that the OD600 of the cells treated with arabinose plateaued at approximately 2.5h, indicating cell growth arrest (Figure 1). Interestingly, both cells treated with 0.2% and 0.4% arabinose demonstrated a resumption in growth at about 12.5h. It is unlikely to be that of BBa_K3198002’s cognate antitoxin, since the transformed plasmid does not contain any antitoxin gene and RES/Xre is orthologous to E. coli. As such, the team hypothesized that either arabinose has a positive effect on cell growth, or that BBa_K3198002 has a relatively short half life and it degrades after approximately 11.5h to result in growth resumption.



Figure 1: Professor Brodersen’s MG1655 containing RES plasmid treated with 0.2% and 0.4% arabinose respectively at 1h.

To test this hypothesis, BBa_K3198002 was cloned under an IPTG-inducible promoter and 2mM IPTG concentration was explored to determine their effect on the growth of native MG1655, using the same characterization conditions and set-up.

Similarly, the cells induced with 2mM IPTG showed a plateau in OD600 almost immediately after induction (Figure 2). However, the automatic resumption in growth remained consistent even when BBa_K3198002 was cloned under an IPTG-inducible promoter. It is also noteworthy that the resumption was earlier in this genetic circuit as compared to Professor Brodersen’s original plasmid.



Figure 2: MG1655 containing RES and Xre plasmids treated with 2mM IPTG at 1h.

Taken together, these results validated BBa_K3198002 ability to inhibit cell growth, but for a relatively short period of time when controlled by both arabinose- and IPTG-inducible systems.

References

Skjerning, R. B., Senissar, M., Winther, K. S., Gerdes, K., & Brodersen, D. E. (2018). The RES domain toxins of RES-Xre toxin-antitoxin modules induce cell stasis by degrading NAD . Molecular Microbiology, 111(1), 221–236. doi: 10.1111/mmi.14150

Milunovic, B., diCenzo, G.C., Morton, R.A.and Finan, T.M. (2014) Cell growth inhibition upon deletion of four toxin‐antitoxin loci from the megaplasmids of Sinorhizobium meliloti. Journal of Bacteriology, 196, 811–824.

Source

BBa_K3198002 was generously provided by Professor Ditlev Brodersen from Aarhus University.

Design Considerations